Tobacco smoke and alcohol, alone or in combination, are associated with an increased risk of cancers including head and neck cancers and lung cancer. Tobacco smoke contains formaldehyde. The metabolism of alcohol releases acetaldehyde. These aldehydes can form DNA-protein and DNA-DNA crosslink adducts, induce genomic instability, and promote cancer. The repair of these DNA crosslink adducts is an important defense against damage from tobacco smoke and alcohol. Mutations in the genes involved in repair of these lesions may be associated with tobacco and alcohol-related cancers. HELQ is a candidate gene to investigate for two reasons: 1) HELQ is involved in DNA crosslink repair. We found that disruption of HELQ in human cells enhances chromosome aberration and cellular sensitivity when exposed to DNA crosslink-inducing agents, and that HELQ associates with the Fanconi anemia (FA) proteins FANCO (RAD51C) and FANCU (XRCC2). The FA pathway is crucial for DNA crosslink repair; and 2) Two independent genome-wide association studies pinpointed the same polymorphism in HELQ as one of the top hits associated with head and neck cancers and lung cancer, particularly amongst heavy alcohol drinkers and tobacco smokers. Further, we have found that one point mutation found in lung cancer is a ubiquitin modification site, and that it influences subcellular localization. However, it is not known whether HELQ processes DNA crosslink damage in the FA pathway. It is also not known whether cancer-associated alterations influence HELQ activity and cellular sensitivity to DNA crosslink-inducing agents. Our central hypothesis is that HELQ functions in the FA pathway and that some mutations and polymorphisms in the HELQ gene alter crosslink repair, leading to increased susceptibility to tobacco and alcohol-related cancers. Guided by preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify effects of HELQ alterations on chromosome aberration and cellular sensitivity upon exposure to crosslink-inducing agents, and the genetic relationship of HELQ with the FA pathway; and 2) Determine biochemical activity of HELQ derivatives on DNA crosslink adducts. Under the first aim, an already proven RNA interference (RNAi) approach will be used to deplete FA genes in our established HELQ knockout human cell lines. Under the second aim, we have purified wild-type HELQ and are ready to generate cancer- associated HELQ variants. The approach is innovative, because HELQ is a newly identified DNA crosslink repair protein and expected outcomes for our proposed studies will identify previously unexplored aspects of DNA crosslink repair in human cells. The proposed research is significant, because once it is known how HELQ and its alterations influence crosslink repair, specific HELQ mutations and polymorphisms can be developed as biomarkers, resulting in innovative risk assessment approaches to the prevention of tobacco and alcohol-related cancers.